FIGURE 3.

SUMOylation of SAE2 C terminus is required for nuclear accumulation of SAE2. A, schematic illustrations of the mutants used for the study (WT construct shown for comparison). B, plasmids shown in A were transiently transfected into HEK239T with or without SAE1, which produced similar results, followed by confocal imaging. SUMOylation of the Cys domain (Cys-K/R) had no effect on SAE2 (top). Conservative mutations that abolished all of the C-terminal SUMOylation sites (Cterm-5K/R) resulted in SAE2 localizing almost completely to the cytoplasm, similar to the deletion of the SAE2 C terminus (Δ575–640, Fig. 2). Transfection and expression of the de-SUMOylation enzyme SENP1 prior to transfection with WT SAE2 induced partial localization of SAE2 to the cytoplasm. Mutations that eliminated the SUMOylation sites outside of NLS (K617/K630 to R; Cterm-2K/R), caused partial localization of SAE2 to the cytoplasm. Mutations that eliminated the SUMOylation sites within the SAE2 NLS (K611,/K613/K623 to R; Cterm-3K/R), also caused a partial localization of SAE2 to the cytoplasm. C, statistical analysis of the localization of SAE2 mutants. The y axis is the ratio of cell with the phenotype divided by the total number of cells. Nuc: found in nucleus only, Nuc+Cyt: found in both nucleus and cytoplasm, and Cyt: found in cytoplasm only. We counted 74–300 cells for each construct from multiple experiments to obtain the averages and standard deviations.